Living cells contain various types of RNA, although three particular types make up the predominant proportion, namely ribosomal RNA (rRNA, approximately 80 percent of all RNAs in a cell), transfer RNA (tRNA, approximately 5 percent of all RNAs in a cell) and messenger RNA (mRNA, approximately 2-4 percent of all RNAs in a cell). A variety of other RNA types are known, including heterogeneous nuclear RNA (hnRNA), small cytoplasmic RNA (scRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), regulatory RNAs, guide RNAs, telomerase RNA and small regulatory RNAs. Isolation of total RNA is typically designed to isolate substantially all of the three predominate types of RNA, that is rRNA, tRNA and mRNA, although minor amounts of the remaining RNA types can be present in the total RNA isolation.
High quality RNA is a prerequisite for various gene expression assays such as reverse transcriptase (RT) PCR, primer extension and Southern or Northern blotting techniques, as well as for preparation of RNA probes for these and similar techniques. In addition novel drug therapies require examining high quality RNA from many species of microbial pathogens. Methods of extracting RNA are known, but those methods have various undesirable characteristics.
Guanidinium thiocyanate and guanidinium chloride are known as effective protein denaturants, and the chloride salt has been employed as a deproteinization agent for isolating total RNA, see Cox, R. A.: Methods in Enzymology, 12(B): 120-129 (1968). In the Cox procedure, a suspension of ribosomes in a buffer is added to 6 molar guanidinium chloride. The RNA is precipitated by adding alcohol to the solution and the precipitate is recovered by centrifugation. At this point, the precipitate is not free of RNase (a contaminating enzyme that causes RNA degradation) and is purified by dissolution in 4 molar guanidinium chloride and re-precipitation by adding ethanol. The precipitate is again separated by centrifugation. If further purification is necessary, the precipitate is redissolved in guanidinium chloride and re-precipitated with ethanol. This procedure takes approximately two days to complete.
Another method for isolating total RNA is disclosed in Chirgwin, J. M. et al: Biochem, 18:5294-5299 (1979). In that procedure, RNA-containing tissue is homogenized in a solution containing guanidinium thiocyanate, sodium citrate and 2-mercaptoethanol, with the solution pH adjusted to 7 by adding sodium hydroxide. The homogenate is then centrifuged and the supernatant decanted and mixed with acetic acid (to lower the pH value to 5) and absolute ethanol. Overnight storage at −20° C. precipitates the RNA and it is recovered in pellet form after centrifugation. The pellet is redissolved in a buffered guanidine hydrochloride solution and reprecipitated by adding acetic acid and ethanol. The last step is repeated and the isolated RNA is recovered in pellet form.
Alternatively, the RNA can be separated from the guanidinium thiocyanate homogenate by ultracentrifugation through a dense cushion of cesium chloride. This method is disadvantageous in that it can take up to 2 days to obtain results and it can require the use of expensive ultracentrifugation equipment which reduces the total amount of RNA recoverable because of the limited number of samples that can be simultaneously processed.
Yet another RNA-isolating procedure is shown in Feramisco, J. R. et al: Molecular Cloning, 194-195, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982), in which RNA-containing tissue is homogenized in a solution of 4M guanidinium isothiocyanate, 20% Sarkosyl™ (sodium lauryl sarkosinate) and 2-mercaptoethanol. An equal volume of heated phenol (approximately 60° C.) is added to the homogenate along with a sodium acetate solution of pH 5.2. Then an equal volume of chloroform is added and the mixture is cooled and centrifuged. The aqueous phase is recovered and reextracted with phenol and/or chloroform about seven more times before the final product is recovered. This procedure is slow and cumbersome in that multiple extractions are required and it takes approximately two days to recover the available RNA.
In U.S. Pat. No. 4,843,155, phenol and guanidine procedures were combined, resulting in a method of total RNA isolation that can be completed in 3 hours. The method of that patent was further improved upon in U.S. Pat. No. 5,346,994, which allows for completion of the RNA isolation in about 1 hour. Those methods involve the use of a mono-phasic solution of phenol and guanidine isothiocyanate, commercially available as the reagent Trizol® (Invitrogen Corp., Carlsbad, Calif.).
It has been found that total RNA samples extracted from clinical isolates using Trizol® or hot phenol methods can experience unacceptable levels of high molecular weight DNA contamination, as determined by ethidium bromide gel electrophoresis. That DNA contamination compromises the purity of the isolated RNA, making subsequent use of the RNA difficult or impossible. Unexpectedly, the methods and compositions of the present invention permit the easy preparation of highly pure RNA samples from clinical isolates with a minimum amount of contaminating genomic DNA. In addition, the methods and compositions of the present invention provide for higher yields and higher purity of total RNA isolated, when compared to other methods.